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Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

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Overexpression or Knockdown of ClC-3 Promotes or Degrades Metastasis in Animal Models of Experimental Pulmonary and Spontaneous Metastases(A and B) Lung metastasis formation at 8 weeks after the injection of HeLa/PcDNA3.1 (A) or HeLa/ClC-3 cells (B) into the tail vein of nude mice. (C and D) Macroscopic aspects of axillary lymph node metastases at about 1-cm xenograft formation after the subcutaneous injection of HeLa/PcDNA3.1 (C) or HeLa/ClC-3 (D) cells into nude mice. (E) Histograms depicting the microscopic or macroscopic metastastic rate in each group are shown. Data are mean ± SEM. ** P < 0.01 vs corresponding control groups. (F-H) Bioluminescence (left) and autopsy (right) imaging after liver orthotopic implantation of high-metastatic potential MHCC97H cells infected with the negative control (GFP alone, shNC, F) or siRNA lentiviruses (ClC-3-siRNA1-GFP, shClC-3, G). Macroscopic metastastic rate in each group are presented in (H). Data are mean ± SEM. (I) Overexpression of ClC-3 in MMTV-PyMT mice (spontaneous mammary tumor model) by crossing with ClC-3 transgenic mice accelerates lung metastasis. Representative lungs from MMTV-PyMT/ClC-3 transgenic mice were isolated at 12 or 14 weeks and stained with Bouin's solution (upper). The number of lungs with metastases was determined using dissecting microscopy and histological examination of H&E-stained sections. The percentage of mice with metastases developed in lung at different phase is shown.
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Figure 2: Overexpression or Knockdown of ClC-3 Promotes or Degrades Metastasis in Animal Models of Experimental Pulmonary and Spontaneous Metastases(A and B) Lung metastasis formation at 8 weeks after the injection of HeLa/PcDNA3.1 (A) or HeLa/ClC-3 cells (B) into the tail vein of nude mice. (C and D) Macroscopic aspects of axillary lymph node metastases at about 1-cm xenograft formation after the subcutaneous injection of HeLa/PcDNA3.1 (C) or HeLa/ClC-3 (D) cells into nude mice. (E) Histograms depicting the microscopic or macroscopic metastastic rate in each group are shown. Data are mean ± SEM. ** P < 0.01 vs corresponding control groups. (F-H) Bioluminescence (left) and autopsy (right) imaging after liver orthotopic implantation of high-metastatic potential MHCC97H cells infected with the negative control (GFP alone, shNC, F) or siRNA lentiviruses (ClC-3-siRNA1-GFP, shClC-3, G). Macroscopic metastastic rate in each group are presented in (H). Data are mean ± SEM. (I) Overexpression of ClC-3 in MMTV-PyMT mice (spontaneous mammary tumor model) by crossing with ClC-3 transgenic mice accelerates lung metastasis. Representative lungs from MMTV-PyMT/ClC-3 transgenic mice were isolated at 12 or 14 weeks and stained with Bouin's solution (upper). The number of lungs with metastases was determined using dissecting microscopy and histological examination of H&E-stained sections. The percentage of mice with metastases developed in lung at different phase is shown.

Mentions: We asked whether ClC-3 function is required during metastasis in a mouse model. There was a low incidence of metastasis with few lung tumor nodules in mice inoculated intravenously with HeLa cells (Figure 2A). However, overexpression of ClC-3 in the HeLa cell line increased lung tumor burden as compared with the HeLa vector cells (Figure 2A, B and 3C). Similarly, up-regulation of ClC-3 expression markedly increased the incidence of lymph node metastasis compared with control stable HeLa cells in the xenograft mouse model (Figure 2C-E). We next investigated the effect of ClC-3 expression knockdown on the lung metastasis potential of high metastatic potential MHCC97H cells. The results demonstrated that there was about 54.5% lung metastasis incidence when MHCC97H cells were embedded in situ into liver. Down-regulation of ClC-3 expression significantly decreased the incidence of metastasis and number of lung tumor nodules (Figure 2F-H and S3C).


Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

Overexpression or Knockdown of ClC-3 Promotes or Degrades Metastasis in Animal Models of Experimental Pulmonary and Spontaneous Metastases(A and B) Lung metastasis formation at 8 weeks after the injection of HeLa/PcDNA3.1 (A) or HeLa/ClC-3 cells (B) into the tail vein of nude mice. (C and D) Macroscopic aspects of axillary lymph node metastases at about 1-cm xenograft formation after the subcutaneous injection of HeLa/PcDNA3.1 (C) or HeLa/ClC-3 (D) cells into nude mice. (E) Histograms depicting the microscopic or macroscopic metastastic rate in each group are shown. Data are mean ± SEM. ** P < 0.01 vs corresponding control groups. (F-H) Bioluminescence (left) and autopsy (right) imaging after liver orthotopic implantation of high-metastatic potential MHCC97H cells infected with the negative control (GFP alone, shNC, F) or siRNA lentiviruses (ClC-3-siRNA1-GFP, shClC-3, G). Macroscopic metastastic rate in each group are presented in (H). Data are mean ± SEM. (I) Overexpression of ClC-3 in MMTV-PyMT mice (spontaneous mammary tumor model) by crossing with ClC-3 transgenic mice accelerates lung metastasis. Representative lungs from MMTV-PyMT/ClC-3 transgenic mice were isolated at 12 or 14 weeks and stained with Bouin's solution (upper). The number of lungs with metastases was determined using dissecting microscopy and histological examination of H&E-stained sections. The percentage of mice with metastases developed in lung at different phase is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385862&req=5

Figure 2: Overexpression or Knockdown of ClC-3 Promotes or Degrades Metastasis in Animal Models of Experimental Pulmonary and Spontaneous Metastases(A and B) Lung metastasis formation at 8 weeks after the injection of HeLa/PcDNA3.1 (A) or HeLa/ClC-3 cells (B) into the tail vein of nude mice. (C and D) Macroscopic aspects of axillary lymph node metastases at about 1-cm xenograft formation after the subcutaneous injection of HeLa/PcDNA3.1 (C) or HeLa/ClC-3 (D) cells into nude mice. (E) Histograms depicting the microscopic or macroscopic metastastic rate in each group are shown. Data are mean ± SEM. ** P < 0.01 vs corresponding control groups. (F-H) Bioluminescence (left) and autopsy (right) imaging after liver orthotopic implantation of high-metastatic potential MHCC97H cells infected with the negative control (GFP alone, shNC, F) or siRNA lentiviruses (ClC-3-siRNA1-GFP, shClC-3, G). Macroscopic metastastic rate in each group are presented in (H). Data are mean ± SEM. (I) Overexpression of ClC-3 in MMTV-PyMT mice (spontaneous mammary tumor model) by crossing with ClC-3 transgenic mice accelerates lung metastasis. Representative lungs from MMTV-PyMT/ClC-3 transgenic mice were isolated at 12 or 14 weeks and stained with Bouin's solution (upper). The number of lungs with metastases was determined using dissecting microscopy and histological examination of H&E-stained sections. The percentage of mice with metastases developed in lung at different phase is shown.
Mentions: We asked whether ClC-3 function is required during metastasis in a mouse model. There was a low incidence of metastasis with few lung tumor nodules in mice inoculated intravenously with HeLa cells (Figure 2A). However, overexpression of ClC-3 in the HeLa cell line increased lung tumor burden as compared with the HeLa vector cells (Figure 2A, B and 3C). Similarly, up-regulation of ClC-3 expression markedly increased the incidence of lymph node metastasis compared with control stable HeLa cells in the xenograft mouse model (Figure 2C-E). We next investigated the effect of ClC-3 expression knockdown on the lung metastasis potential of high metastatic potential MHCC97H cells. The results demonstrated that there was about 54.5% lung metastasis incidence when MHCC97H cells were embedded in situ into liver. Down-regulation of ClC-3 expression significantly decreased the incidence of metastasis and number of lung tumor nodules (Figure 2F-H and S3C).

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Show MeSH
Related in: MedlinePlus